The specific gravity of a test sample, such as urine or serum, is a measure of the relative proportions of solid material dissolved in the test sample to the total volume of the test sample. In general, the specific gravity of a test sample is a measure of the relative degree of concentration or the relative degree of dilution of the test sample. The specific gravity of urine can be correlated to the cation concentration, and especially the divalent cation concentration of the urine sample. With regard to urine samples, the assay for specific gravity helps interpret the results of the other assays performed in a routine urinalysis.
Clinically, under appropriate and standardized conditions of fluid restriction or increased fluid intake, the specific gravity of a urine sample measures the concentrating and diluting abilities of the kidneys of an individual. The specific gravity of urine ranges from about 1.005 to about 1.030, and usually is in the range from about 1.010 to about 1.025. A specific gravity of about 1.025 or above in a random first morning urine specimen indicates a normal concentrating ability of the kidneys.
Either an abnormally low or an abnormally high urine specific gravity is clinically significant. Therefore, accurate and reliable specific gravity assays of urine and other aqueous test samples must be available for both laboratory and home use. The assays must permit the accurate measurement of abnormally low and abnormally high specific gravities, such that a correct diagnosis can be made and correct medical treatment implemented, monitored and maintained.
For example, diabetes insipidus, a disease caused by the absence of, or impairment to, the normal functioning of the antidiuretic hormone (ADH), is the most severe example of impaired kidney concentrating ability. This disease is characterized by excreting large urine volumes of low specific gravity. The urine specific gravity of individuals suffering diabetes insipidus usually ranges between 1.001 and 1.003. Low urine specific gravity also occurs in persons suffering from glomerulonephritis, pyelonephritis, and various other renal anomalies. In these cases, the kidney has lost its ability to concentrate the urine because of tubular damage.
An abnormally high urine specific gravity also is indicative of a diseased state. For example, the urine specific gravity is abnormally high in an individual suffering from diabetes mellitus, adrenal insufficiency, hepatic disease or congestive cardiac failure. Urine specific gravity likewise is elevated when an individual has lost an excessive amount of water, such as with sweating, fever, vomiting, and diarrhea. In addition, abnormally high amounts of nonionic urinary constituents, like glucose and protein, increase the urine specific gravity to 1.050 or greater in some individuals suffering from diabetes mellitus or nephrosis. Furthermore, urine with a fixed low specific gravity of approximately 1.010 that varies little from specimen to specimen is known as isothenuric. This condition is indicative of severe renal damage with disturbance of both the concentrating and diluting abilities of the kidney.
Therefore, in order to determine if an individual has either an abnormally high or an abnormally low urine specific gravity, and in order to help monitor the course of a medical treatment to determine its effectiveness, simple, accurate and inexpensive specific gravity assays have been developed. In general, the specific gravity of a test sample is a measurement that relates to the density of the test sample. The specific gravity is a value derived from the ratio of the weight of a given volume of a test sample, such as urine, to the weight of the same volume of water under standardized conditions (Eq. 1). ##EQU1## Water has a specific gravity of 1.000. Since urine is a solution of minerals, salts, and organic compounds in water, the specific gravity of urine is greater than 1.000. The relative difference reflects the degree of concentration of the urine specimen and is a measure of the total solids in urine.
Several methods are available to determine the specific gravity of urine. The most widely used method, and possibly the least accurate, employs a urinometer. The urinometer is a weighted, bulb-shaped instrument having a cylindrical stem containing a scale calibrated in specific gravity readings. The urinometer is floated in a cylinder containing the urine sample, and the specific gravity of the urine is determined by the depth the urinometer sinks in the urine sample. The specific gravity value is read directly from the urinometer scale at the junction of the urine with the air. The urinometer method is cumbersome and suffers from the disadvantages of a) requiring large volumes of urine test sample, b) a difficult and inaccurate reading of the urinometer scale, and c) unreliable assays because the urinometer is not regularly recalibrated.
Refractometry provides an indirect method of measuring the specific gravity of urine. The refractive index of urine is directly related to the number of dissolved particles in urine and, therefore, is directly related to the specific gravity of urine. Consequently, measurement of the refractive index of urine can be correlated to the specific gravity of urine. The refractometer method of determining urine specific gravity is desirable because specific gravity measurements are performed on as little as one drop of urine. However, the refractometer has the disadvantages of requiring daily calibration and not being amenable to home assays.
A third urinalysis method for specific gravity, the falling drop method, like the urinometer, is a direct measurement of urine specific gravity. In this method, a drop of urine is introduced into each of a series of columns filled with solvent mixtures of increasing and known specific gravity. When the drop of urine comes to rest after its initial momentum has dissipitated, and then neither rises nor falls, the specific gravity of the urine is determined to be identical to the specific gravity of the solvent mixture of that particular column. The falling drop method, however, is not widely used in routine urinalysis because of the lengthy time requirements in setting up such a assay and the inability of an individual to perform the assay at home.
The falling drop method described above also can be performed instrumentally. The instrument-based assay uses a specially designed column filled with a silicone oil having a controlled specific gravity and viscosity. The column is designed to measure the time required for a precisely measured drop of test sample to fall a distance defined by two optical gates (lamp-phototransistor pairs) mounted one above the other in a temperature-controlled column filled with a water-immiscible silicone oil of a slightly lower density than the test sample. The falling time is measured electronically and computed into specific gravity units. This specific gravity method is very precise, however, the cost of the assay instrument and the degree of skill required to operate the instrument makes home testing for urine specific gravity impractical.
Not one of the above-described specific gravity assay methods is suited to performing specific gravity assays outside a medical office or laboratory. Consequently, reagent impregnated test strips were developed to perform specific gravity assays at home. In general, the test strip assay developed for specific gravity determinations is an indirect assay method, wherein the test strip changes color in response to the ionic strength of the urine sample.
The present day specific gravity test strips comprise a carrier matrix impregnated with a reagent composition including a polyelectrolyte, such as a partially neutralized poly(methyl vinyl ether/maleic acid); a chromogenic indicator, such as bromothymol blue; and suitable buffering agents. The reagent composition is sensitive to the number of ions, or electrolytes, in the urine sample, such that the polyelectrolyte of the reagent composition undergoes a pK.sub.a (acid dissociation constant) change in relation to the ionic strength of the urine sample. Therefore, as the concentration of electrolytes in the urine increases (high specific gravity), the pK.sub.a of the polyelectrolyte present in the reagent composition decreases because free carboxyl groups are converted to carboxylate groups. The overall result is a pH decrease and a color transition of the bromothymol blue chromogenic indicator from blue-green to green to yellow-green in response to increased specific gravity. The resulting color transition, indicating a pH change caused by increasing ionic strength, i.e., increasing specific gravity, is empirically related to the specific gravity of the test sample.
For test strips utilizing the partially neutralized poly(methyl vinyl ether/maleic acid) polyelectrolyte and bromothymol blue indicator, assays for specific gravity are performed on aqueous test samples having a specific gravity ranging from 1.000 to 1.030. A reading of 1.000, or a blue-green color, indicates that the urine has a very low specific gravity, as demonstrated by the lack of a color transition of the chromogenic indicator dye. A specific gravity reading of from 1.005 to 1.030 is signified by color transitions, of from blue-green through green to yellow-green, that serve as reliable indicators of increasing specific gravity.
It would be extremely advantageous to have a simple and trustworthy method of semiquantitatively assaying for urine specific gravity that allows visual differentiation of specific gravity values within the range of 1.000 to about 1.050. By providing a semiquantitative method of determining urine specific gravity in an easy to use form, such as a dip-and-read test strip, the urine assay can be performed by laboratory personnel to afford immediate test results. The specific gravity assay results can be interpreted in conjunction with assays for other urine constituents, such that a diagnosis can be made without having to wait for assay results and medical treatment can be commenced immediately. Furthermore, the test strip method can be performed by an individual at home to estimate the specific gravity of the urine and therefore to help monitor the success of the medical treatment the individual is undergoing.
As will be described more fully hereinafter, the method of the present invention allows the fast and trustworthy assay for the divalent cation concentration or the specific gravity of urine by utilizing a test strip having a test pad that incorporates a reagent composition comprising a metal-sensitive triphenylmethane (MSTPM) dye. The reagent composition undergoes a color transition in response to the divalent cation concentration of the test sample. The color transition is directly related to the divalent metal ion concentration. Therefore, the reagent composition including the MSTPM dye provides sufficient assay sensitivity to allow the quantitative determination of divalent cation concentration and the semiquantitative determination of specific gravity.
Any method of assaying for the divalent cation concentration or the specific gravity of urine or other aqueous test samples must yield trustworthy and reproducible results by utilizing a reagent composition that undergoes a color transition due to an interaction in response to the divalent cation concentration or to the specific gravity of the test sample, and not as a result of a competing chemical or physical interaction, such as a pH change or preferential interaction with another test sample component, like protein or glucose. Additionally, the method and composition utilized in the divalent cation assay or specific gravity assay should not adversely affect or interfere with the other test reagent pads that are present on multiple test pad strips.
In accordance with the present invention, a reagent composition incorporated into the carrier matrix significantly reduces the development of an interfering background color, and thereby provides a sufficient sensitivity and color differentiation to assay for divalent cation concentration quantitatively, or for specific gravity semiquantitatively, especially in the range of about 1.000 to about 1.015. In addition, although dry phase test strips have been used to assay for specific gravity, no dry phase test strip has incorporated an MSTPM dye to provide sufficient sensitivity and sufficient visual color resolution to allow the assay of divalent cation concentration, or to allow the semiquantitative specific gravity assay of a test sample.
The prior art contains references to the polyelectrolyte-dye chemistry utilized in the above-discussed specific gravity assay of urine. For example, Falb et al. U.S. Pat. No. 4,318,709 and Stiso et al. U.S. Pat. No. 4,376,827 disclose the basic polyelectrolyte-dye technique used to assay for urine specific gravity. Both patents teach utilizing polyelectrolyte-dye chemistry to determine the specific gravity of urine by monitoring the color transition of the dye.
However, the present invention provides a composition and method for the accurate determination of divalent cation concentration, or the semiquantitative determination of specific gravity, of urine and other aqueous test samples by utilizing an MSTPM dye as the indicator component of a reagent composition in the absence of a polyelectrolyte. European Patent Application 0 349 934 discloses a test strip and method of determining specific gravity or ionic strength of a sample utilizing a composition including a buffer, a complex former and a pH indicator dye. The complex former can be a crown ether, a cryptand, a podand or a multifunctional liquid. The pH indicator dye is a standard dye, such as bromothymol blue or thymol blue. European Patent Application 0 349 934 does not teach or suggest an MSTPM dye utilized in the present invention.
Greyson et al., in U.S. Pat. No. 4,015,462, disclose a support matrix incorporating osmotically-friable microcapsules containing a fluid including a dye. A portion of the microcapsules bursts upon contact with a test sample of low osmolality. A resulting release of the dye-containing fluid causes a color transition that is correlated to the specific gravity. However, the difficult production of the microencapsulated-containing supporting matrix is a serious disadvantage of the Greyson et al. method.
In contrast to the prior art, and in contrast to the presently available commercial test strips, the method of the present invention provides increased sensitivity in the measurement of urine divalent cation concentration, and provides a semiquantitative measurement of urine specific gravity, by utilizing a reagent composition including a MSTPM dye, a buffer, and optionally a chelating agent, wherein the reagent composition is essentially free of polyelectrolytes. The present reagent composition effectively reduces the development of a background color in the test pad thereby either providing a semiquantitative specific gravity assay, or providing an accurate determination of divalent cation concentration. Hence, new and unexpected results are achieved in the dry phase reagent strip assay of urine and other aqueous test samples for divalent cation concentration or for specific gravity.